Platelet- and plasma-derived factor Va serve an essential role in thrombin generation catalyzed by Prothrombinase, which consists of 1:1, stoichiometric, calcium-dependent complex of the cofactor factor Va and the serine protease factor Xa bound to activated platelets. Removal of factor Va from the complex results in a 10,000-fold decrease in the rate of thrombin generation, the physiologic effect being demonstrated by the severe hemorrhage observed in factor V deficiency. Recent studies indicate that the entire pool of platelet-derived factor V originates from plasma through endocytosis of plasma factor V by platelet progenitor cells, megakaryocytes. However, platelet-derived factor Va exhibits biochemical and physical differences that clearly distinguish it from plasma-derived factor Va, differences that impart an increased procoagulant potential to the platelet-derived cofactor. The goal of this project is to understand the intracellular processes that produce a platelet-derived cofactor molecule that is physically and functionally unique when compared to its plasma counterpart. This proposal details strategies to test the hypothesis that plasma-derived factor V is endocytosed by megakaryocytes, trafficked to the trans-Golgi network, retailored posttranslationally, packaged into alpha-granules and processed proteolytically to yield the entire pool of the platelet-derived cofactor. The first aim is to correlate factor V endocytosis with megakaryocyte differentiation and maturity using megakaryocytes generated by ex vivo expansion of CD34+ bone marrow cells, primary bone marrow-derived megakaryocytes, and appropriate megakaryocyte-like cell lines. The second aim is to define the cellular events that regulate factor V endocytosis, its intracellular trafficking to alpha-granules (perhaps through the trans-Golgi network), and the phenotypic changes in factor V resulting from these interactions. Since platelet-derived factor Va plays a more essential role in maintaining normal hemostasis than does its plasma counterpart, these studies will increase our understanding of how megakaryocytes acquire, process and package this critical coagulation factor.